Creation of a resistant KMS-12-BM-BPR sub-line. To develop a Btz-resistant cell line using clinically relevant pulse-treatment, we treated KMS-12-BM cells twice weekly with 1hr pulses of Btz. As resistance developed, we increased the dose stepwise from 900nM to 7.2µM Btz (Fig. 1a) over the course of six months, until we derived a sub-line that was approximately 10-fold more resistant (Fig. 1b). We named it KMS-12-BM-BPR, where BPR stands for “bortezomib-pulse resistant”. For brevity, we will also refer to it as BPR. This sub-line did not require culturing in the presence of Btz to maintain resistance and grew at the same rate as the parental line (Fig. S1a). Thus, it is feasible to develop Btz-resistant cell lines using clinically relevant pulse treatments.
KMS-12-BPR cells line does not have mutations and does not overexpress proteasomes. We tested whether the BPR cells have a mutation in or around Btz binding pocket of the PSMB5 (β5c) subunit, which reduced Btz affinity to its prime target, as in the majority of previously developed Btz-resistant cells9–11. We isolated genomic DNA from both cells and Sanger-sequenced the exomes of PSMB5, PSMB6 (β1c), and PSMB7 (β2c) genes that encode catalytic subunits of the constitutive proteasome, and the PSMB8 (β5i), PSMB9 (β1i), PSMB10 (β2i) genes that encode catalytic subunits of lymphoid tissue specific immunoproteasome, which are expressed in the myeloma cells21–23. We did not find any specific mutations in the resistant cells. Some of previously reported resistant cells overexpressed the β5 subunits9, resulting in an increase in proteasome activity. Although we observed a small increase in the chymotrypsin-like activity in the resistant cells, it was not significant (Fig. 1c).
Consistent with a lack of mutations, 1hr pulse treatment with Btz caused similar inhibition of ß5 (i.e., combined activity of β5c and β5i) and ß1 (i.e., combined activity of β1c and β1i) activities in the parental cells and the resistant subclone (Fig. 1d). Although there were small differences in inhibition of β5 activity at sub-toxic Btz concentrations, inhibition was the same in cells treated with 1µM Btz, which causes largest differences in viability. Thus, resistance in the BPR sub-line is not due to decreased affinity of Btz to the active sites. These data also indicate that the drug is still penetrating the cell and, most likely, is not being transported out of the cell at an increased rate.
Faster recovery of the proteasome activity after pulse treatment could contribute to resistance. We measured the proteasome activity of the cells over time after a pulse treatment with 1µM Btz, which causes maximal differences in sensitivity (Fig. 1d). The resistant cells have a slightly faster recovery of the β5 activity, but not the β1 activity. 20% of β5 activity recovered 4-6h after treatment in resistant cells (Fig. 1e), which is before the onset of apoptosis (Fig. 1f) raising the possibility that differences in recovery rates may contribute to resistance.
To determine whether different recovery rates translate into differences in protein degradation, we measured degradation of long-lived proteins in a pulse-chase experiment and accumulation of undegraded K48-linked ubiquitylated proteins, most of which are derived from undegraded nascent polypeptides24. After 1hr treatment, both approaches revealed a modest difference between sensitive and resistant cell lines, with resistant cells being slightly less susceptible for inhibition (Fig. 1g). Despite modest initial difference, sensitive cells accumulated substantially more ubiquitylated proteins over time. The difference was notable at 6hr and preceded the onset of apoptosis, which was detected only at 12hr (Fig. 1h), and therefore is more likely to be the cause and not the consequence of cell death. The decreased accumulation of ubiquitylated proteins in resistant cells could be caused by activation of deubiquitylating enzymes. However, we did not detect any activation of deubiquitylating enzymes using the ubiquitin-methyl ester activity-based probe25 (Fig. S1b). Therefore, difference in the degradation rates is the most likely cause of resistance. How can similar occupancy of active sites by Btz lead to differences in protein degradation rates that appear to increase over time?
Sensitivity of myeloma cells to proteasome inhibitors is defined by the ratio of the load on the proteasome, i.e. amount of proteins degraded, to proteasome capacity, i.e. amount of active proteasomes1–5. Cells with lower load have many idling proteasomes and can maintain rates of protein degradation when proteasomes are partially inhibited (e.g., β5 sites are inhibited but two other sites are functional) simply by engaging idling proteasomes. Increasing load-to-capacity will engage idling proteasome and reduce spare capacity. Under this condition, even partial inhibition of individual active sites will result in the inhibition of protein degradation, resulting in accumulation of ubiquitylated proteins at lower concentrations of Btz, ultimately leading to higher Btz sensitivity. Since proteasome activity in the resistant cells was similar (Fig. 1c), increased capacity cannot account for resistance, suggesting that lower load is the major cause of resistance. The majority of ubiquitylated proteins that accumulate upon treatment of cells with proteasome inhibitor are derived from nascent polypeptides24, therefore higher rates of protein synthesis should result in the higher load on the proteasome. We used puromycin incorporation assay26 to measure rates of protein synthesis, and found that resistant cells incorporate substantially less puromycin that the sensitive one indicating slower protein synthesis rates (Fig. 1h). Thus, Btz resistance in KMS-12-BM-BPR cells is caused by decreased load on the proteasome due to lower protein synthesis rate.
Cfz and marizomib (Mzb), but not Ixz overcome Btz resistance. We next tested if other proteasome inhibitors could overcome Btz resistance in KMS-12-BM-BPR line (Fig. 2). We observed that Cfz, which is approved by the FDA for the treatment of multiple myeloma, and marizomib (Mzb, salinosporamide A, MPI-0052), a natural product undergoing clinical trials in multiple myeloma and glioblastoma, can overcome resistance (Fig. 2a). However, Ixz, an orally bioavailable FDA-approved analogue of Btz, did not. Unlike Cfz and Mzb, Btz and Ixz do not inhibit proteasome β2 sites, responsible for the trypsin-like activity. Although all four inhibitors blocked β5 with similar potency, we found that decrease of viability in Cfz and Mzb-treated cells coincided with co-inhibition of ß1 and ß2 sites (Fig. 2b). In comparison, Ixa and Btz inhibit ß2 activity at higher concentrations than ß1 (Fig. 2b and 2c). Thus, it appears that inhibition of β2 sites is critical to overcoming Btz-resistance of KMS-12-BM BPR cells.
Increasing proteasome inhibition by targeting the β2 subunits can overcome Btz resistance. The ability of inhibitors that co-inhibit β2 sites to overcome Btz resistance is consistent with an expectation of the load-to-capacity hypothesis that resistant cells, which are able to withstand partial proteasome inhibition due to their low load-to-capacity ratio, will succumb to proteasome inhibitors that decrease proteasome capacity by blocking additional active sites. We previously found that β2 specific inhibitor LU-102 is a potent sensitizer of myeloma and solid tumor cells to Btz and Cfz, and that it overcomes acquired resistance to these agents23,27−29. To confirm the importance of ß2 sites for Btz resistance in the BPR line, we tested whether LU-102 restores Btz sensitivity. We found that specific blocking of β2 sites by LU-102 had no effect on cell viability of parental and resistant cells (Fig. 3a). When 1hr Btz treatment was followed by a subsequent treatment with sub-toxic, β2 specific concentrations of LU-102, both cell lines were sensitized to Btz (Fig. 3b). However, sensitization was much stronger in resistant cells. Co-treatment with LU-102 sensitized resistant cells to a wider range of Btz concentrations. The effect of LU-102 on viability of resistant cells was most noticeable at 1µM Btz, where addition of LU-102 restored the Btz sensitivity of KMS-12-BM-BPR cells to the level of the wild type. At this concentration, co-treatment with LU-102 accelerated accumulation of ubiquitylated proteins (Fig. 3c), which confirms that LU-102 acts through inhibition of proteasome and rules out contribution of autophagy to resistance. Thus, inhibition of ß2 sites overcomes resistance to Btz in KMS-12-BM-BPR cells.
KMS-12-BM-BPR cells are highly sensitive to Bcl-2 inhibition. We noticed that the resistant cells consistently turned phenol-red containing media orange and yellow (indicative of a pH change) at a faster rate than their parental cells and confirmed increased production of lactate (Fig. 4a), which may indicate reduced mitochondrial respiration. KMS-12-BM cells bear t(11;14) translocation. According to the literature, myeloma cells bearing t(11;14) translocation have lower oxygen consumption rates than myeloma cells without this translocation30, and are more sensitive to Bcl-2 inhibitor ABT-199 (venetoclax)31,32. KMS-12-BM, however, is not the most venetoclax-sensitive myeloma line31. Therefore, we decided to investigate whether KMS-12-BM-BPR are more sensitive to Bcl-2 inhibitors than the parental cells.
We observed an increased sensitivity of the resistant cells to the dual Bcl-2/Bcl-XL inhibitor, ABT-737 (Fig. 4b) and to Bcl-2 specific inhibitor ABT-199 (venetoclax, Fig. 4C) but not to the BCL-XL specific inhibitor A-115546333 (Fig. 4c). When a 1hr Btz treatment was followed by ABT-199, the combination was synergistic in both cell lines (Fig. 4d). Sensitivity of myeloma cells to venetoclax depends on Bcl-2:Mcl-1 and Bcl-2:Bcl-XL expression ratios32. Therefore, to determine the mechanism of increased Bcl-2 dependence, we examined expression of Bcl-2 members and found that Mcl-1 expression was dramatically reduced in the resistant cells, but Bcl-2 was not overexpressed in the resistant cells (Fig. 4e). Bcl-XL expression was low in both lines (Fig. 4e). We conclude that increased Bcl-2:Mcl-1 ratio due to decreased expression of Mcl-1 sensitizes KMS-12-BM-BPR cells to venetoclax.